Clostridium difficile infection (CDI) has become a major emerging cause of morbidity and mortality among hospitalized patients since the epidemics of hypervirulent and antibiotic-resistant strains began in the early 2000s. It is therefore important to develop new preventions/treatments to counteract the rapid increase in the incidence of CDI. While standard treatments with antibiotics are not effective resulting in high rates of relapse, animal and clinical studies have unambiguously demonstrated that antitoxin antibodies are protective against both primary and recurrent CDI. Our goal is to develop novel immunoprophylaxes against both primary and recurrent CDI in aged high-risk populations. In this proposal we aim to exploit two novel compounds (ABBA and cTxAB) developed in this lab: 1) ABBA is a single tetra-specific binding agent consisting of four distinct neutralizing VHH monomers (two against TcdA and two against TcdB); and 2) cTxAB is an atoxic, clostridial toxin-like chimeric vaccine for which a product development plan (PDP) toward clinical trials is currently being developed under NIH funding. We have already demonstrated potent efficacy of ABBA against fulminant CDI in mice. We will further optimize and finalize its delivery strategies in this project. To reduce potential immunogenicity and increase the serum half-life of ABBA for systemic administration, we will generate ABBA-Fc fusions and humanized ABBA-lgGIs without compromising its superior affinity to toxins and neutralizing activity. In addition, we will develop a colonic delivery system for intestinal delivery of ABBA. The lead compounds generated for systemic and oral deliveries will be evaluated in a hamster fulminant CDI model as well as in gnotobiotic piglets colonized with human intestinal flora. Moreover, we will combine the passive ABBA immunization with active cTxAB vaccination and evaluate protection against both primary and recurrent CDI in relevant immunosenescent mouse models. Our ultimate goal is to develop novel immunoprophylaxes and combinations of active and passive immunizations that we wish to prevent against both immediate and longer term CDI threats in aged patients with high risk of developing this debilitating disease. This project will help to achieve the broad objective ofthe entire CETR proposal and to address the hypothesis that we can translate promising immunoprophylactic candidate products being tested in relevant immunosenescent animal models into immune preventions against important enteric pathogens for which the ultimate human targets are elderly subjects.